Functionalization of fatty acid mimetics for solid-phase coupling and subsequent target identification

Article abstract of DOI:10.1002/ardp.201000091

Fatty acid mimetics such as pirinixic acid (PA) derivatives and 2-(phenylthio)alkanoic acid derivatives are drug-like small molecules with an interesting pharmacological profile. Previously, we have characterized PA derivatives (e.g., 1) as dual agonists

Full text of DOI:10.1002/ardp.201000091

Arch. Pharm. Chem. Life Sci. 2010, 10, 625–630
625
Short Communication
Functionalization of Fatty Acid Mimetics for Solid-Phase
Coupling and Subsequent Target Identification
Michaela Dittrich1, Heiko Zettl1,2, and Manfred Schubert-Zsilavecz1
1 Goethe-University Frankfurt, Institute of Pharmaceutical Chemistry, ZAFES/LiFF, Frankfurt, Germany
2 present address: ETH Zurich, Institute of Pharmaceutical Sciences, Zurich, Switzerland
Fatty acid mimetics such as pirinixic acid (PA) derivatives and 2-(phenylthio)alkanoic acid derivatives
are drug-like small molecules with an interesting pharmacological profile. Previously, we have
characterized PA derivatives (e.g., 1) as dual agonists of peroxisome proliferator-activated receptors
(PPARs) a and g and as inhibitors of microsomal prostaglandin E2-synthase-1 (mPGES-1) and 5-
lipoxygenase (5-LO). 2-(Phenylthio)alkanoic acids (e.g., 2) were shown to act as highly active and
selective PPARa agonists. Encouraged by these results, we would like to identify other target
proteins and, thereby, further explore the pharmacological profile of these molecules. An elegant
method to screen for potential interaction partners is the so-called ‘‘protein-fishing’’ approach.
Requirement is coupling of a functionalized small molecule to a solid phase which is used for
biological experiments. Ideally, the pharmacophore of the small molecule remains intact as far as
possible. Here, we describe the successful design and synthesis of functionalized fatty acid mimetics,
thus providing an eligible starting point for solid-phase coupling and subsequent ‘‘protein-fishing’’
experiments.
Keywords: Fatty acid mimetics / Functional groups / Pirinixic acid / Protein fishing / Solid-phase coupling
Received: March 23, 2010; Accepted: May 3, 2010
DOI 10.1002/ardp.201000091
Introduction
treatment of the metabolic diseases dyslipidemia (PPARa
agonists like fenofibrate) and type-2 diabetes mellitus
(PPARg agonists like pioglitazone). Inhibitors of mPGES-1
and 5-LO have shown to exert multiple anti-inflammatory
effects, hence, displaying a promising alternative to classical
non-steroidal anti-inflammatory drugs (NSAIDs), which are
associated with severe side effects such as gastrointestinal
toxicity and increased cardiovascular morbidity [6]. Our
previous studies include systematic structural variations of
the PA scaffold providing detailed information about struc-
ture–activity relationships (SAR). As a result, most active
compounds for PPAR and mPGES-1/5-LO such as 1 (Fig. 2)
contain an n-hexyl chain in the a-position and a biphenyl
residue coupled to the pyrimidine ring [1]. Notably, the
substitution pattern of the biphenyl moiety had only minor
impact on the PPAR activation and the dual mPGES-1/5-LO
inhibition. PA derivative 1 is a PPARa/g dual agonist with an
EC50 value of 0.19 mM for PPARa and 1.5 mM for PPARg [1].
Furthermore, this compound also shows high inhibitory
activities for 5-LO (IC50 ¼ 0.41 mM) and mPGES-1
Fatty acid mimetics like pirinixic acid (PA) and derivatives
(Fig. 1) display an interesting pharmacophore with multiple
biological activities. Previously, we have shown that pirinixic
acid derivatives act as activators of a subclass of nuclear
receptors i.e., peroxisome proliferator-activated receptors
(PPARs)
a and g [1–3], and as inhibitors of distinct
enzymes of the arachidonic acid cascade i.e., microsomal
prostaglandin E2-synthase-1 (mPGES-1) and 5-lipoxygenase
(5-LO) [4, 5]. PPAR agonists are widely used drugs in the
Correspondence: Prof. Dr. Manfred Schubert-Zsilavecz, Institute of
Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-
Str. 9, D-60438 Frankfurt, Germany.
E-mail: Schubert-Zsilavecz@pharmchem.uni-frankfurt.de
Fax: þ49-69-79829332
Abbreviations: 5-lipoxygenase (5-LO); microsomal prostaglandin E2-
synthase-1 (mPGES-1); peroxisome proliferator-activated receptors
(PPARs); pirinixic acid (PA).
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Arch. Pharm. Chem. Life Sci. 2010, 10, 625–630
Figure 1. Scaffold of PA derivatives and 2-(phenylthio)alkanoic acid derivatives [1, 3].
(IC50 ¼ 1.7 mM). Based on these findings, we have selected the
robust scaffold of 1 for the purpose of this study in order to
ensure high pharmacological activity.
To further investigate the pharmacological profile of the
selected fatty acid mimetics (i.e., compounds 1 and 2), we were
looking for a possibility to identify targets other than the
known PPAR, mPGES-1, and 5-LO. An excellent screening
method is the so-called ‘‘protein-fishing’’ approach, which
was applied successfully in many recent research studies
[8–10]. In short, the underlying strategy of this method is
coupling of a functionalized small molecule to a polymer
matrix as solid phase. Structural requirement for successful
solid-phase coupling is the introduction of an eligible func-
tional group (i.e., amine, hydroxyl, or carboxylic acid) to the
initial pharmacophore. Importantly, the position for this
Structural optimization efforts of the PA lead structure
yielded a novel class of 2-(phenylthio)hexanoic acid deriva-
tives (Fig. 1) [4]. Compounds based on this novel scaffold are
selective PPARa agonists with nanomolar activity. SAR
revealed a high structural tolerance to modifications of
the terminal phenyl residue. Encouraged by the promising
PPAR activities of this series, we have selected representative
2
for this work (EC50 PPARa ¼ 0.056 mM and EC50
PPARg ¼ 3.02 mM) [7].
Figure 2. Functionalization of PA derivative 1 and 2-(phenylthio)alkanoic acid derivative 2.
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Arch. Pharm. Chem. Life Sci. 2010, 10, 625–630
Functionalization of Fatty Acid Mimetics
627
chemical modification has to be selected carefully to ensure
that the structural entity remains untouched as far as
possible.
acid derivatives 5a and 5b. Solid-phase coupling will be done
by amidation on a Toyopearl AF-Amino-650 resin [11].
Notably, the ester moiety of 5a and 5b has to be cleaved after
the coupling reaction in order to ensure regioselectivity.
2) For functionalization of the terminal 4-phenyl position
of compound 2, the introduction of a hydroxyl (using 3,4-
dimethylhydroquinone) or an amine (using 4-amino-2,3-xyle-
nol) were possible. Because of the susceptibility to oxidation
This short communication addresses the issue of pharma-
cophore functionalization based on the selected fatty acid
mimetics 1 and 2. Subsequent experiments might be the
incubation of the matrix-small molecule complex with cell
lysates of interest in order to investigate specific ligand–
protein interactions.
of 2,3-dimethylhydroquinone impairing its use in
a
Mitsunobu reaction (Scheme 2, step (iii)), we have decided
to introduce an amine by using 4-amino-2,3-xylenol. Finally,
only the boc-protected 4-amino-2,3-xylenol reacted under
Mitsunobu conditions to give 10a and 10b. Solid-phase
coupling will be done on a Toyopearl AF-Epoxy-650 resin [12].
Synthesis of the pirinixic acid derivatives 5a and 5b is
shown in Scheme 1 and was adapted from d’Atri et al.
[4, 13]. The starting reaction was a nucleophilic substitution
of 2-mercapto-pyrimidine-4,6-diol (2-thiobarbituric acid) with
ethyl 2-bromooctanoate 3a and ethyl 2-bromoacetate 3b,
respectively, in the presence of NEt3 (Scheme 1; step (i)).
Next, chlorination of the hydroxyl groups with phosphorus
oxy chloride afforded the dichloropyrimidine derivatives 4a
and 4b in quantitative yield (Scheme 1; step (ii)). We achieved
the final compounds 5a and 5b by amination with 40-ami-
no(1,10-biphenyl)-4-carboxylic acid in the presence of equi-
molar amounts of NEt3 (Scheme 1; step (iii)) [13].
Results and discussion
As stated in the introduction, we have selected compounds 1
and 2 for functionalization in order to enable solid–phase
coupling (Fig. 2).
Our previous SAR studies showed clearly the importance of
the carboxylic acid head group and the a-alkyl chain for
pharmacological activity of 1 and 2. In contrast, structural
modifications of the lipophilic backbone were rather toler-
ated. Therefore, we have selected the terminal phenyl ring of
both 1 and 2 for the introduction of an additional functional
group (Fig. 2, 5a and 10a). Additionally, we have synthesized
the respective a-unsubstituted derivatives (5b and 10b) to
provide an additional ‘‘internal standard’’ for following bio-
logical experiments. Considering the commercial availability
of respective educts, these synthetic options have the follow-
ing results:
1) For functionalization of the 40-biphenyl position of com-
pound 1, the replacement of the cyano group by an amine
(using biphenyl-4,40-diamine) or by a carboxylic acid (using 40-
amino-biphenyl-4-carboxylic acid) was possible. To avoid a
cross-linking reaction with biphenyl-4,40-diamine in the
amination step (Fig. 2; Scheme 2, step (iii)), we have decided
to use 40-amino-biphenyl-4-carboxylic acid yielding carboxylic
Preparation of the compounds 10a and 10b was adapted
from the recently published synthesis of 2 as shown in
Scheme 2 [7]. First, the esterified acidic head group was
arranged by nucleophilic substitution of 4-mercaptophenol
with ethyl 2-bromohexanoate 6a and ethyl 2-bromoacetate
6b, respectively (Scheme 2; step (i)) [15]. The propylene spacer
was introduced by a Williamson-like ether synthesis with 3-
bromopropan-1-ol to give 7a and 7b (Scheme 2; step (ii)) [16].
Scheme 1. Synthesis of 5a and 5b.
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Arch. Pharm. Chem. Life Sci. 2010, 10, 625–630
Scheme 2. Synthesis of 10a and 10b.
(1.28 mmol, 1 eq) with 0.178 mL of triethylamine (1.28 mmol,
1 eq) in 40 mL EtOH. 4-Amino(biphenyl) carboxylic acid hydro-
chloride was suspended in ethanol. Triethylamine was added
and the mixture was heated to 808C for 1 h. The reaction mixture
was filtered hot and the pure amine was obtained by concen-
trating the filtrate in vacuo (0.161 g, 59%). Next, 0.165 g of 4a
(0.47 mmol, 1 eq) and 0.1 g of 4-amino(biphenyl) carboxylic acid
(0.47 mmol, 1 eq) were dissolved in 40 mL EtOH and 0.065 mL
triethylamine (0.47 mmol, 1 eq) were added. The reaction mix-
ture was heated to 808C for 126 h. After the reaction was com-
pleted (TLC control), all volatile components were removed
in vacuo. The purification by column chromatography (n-hex-
ane/ethyl acetate, 10:1; n-hexane/ethyl acetate, 5:1; n-hexane/ethyl
acetate, 3:1; ethyl acetate) gave a white solid (0.143 g, 58%). 1H-
NMR (300.13 MHz, DMSO-d6) d: 0.78 (t, 3H, J ¼ 6.59 Hz, CH3-Hex),
1.14 (t, 3H, J ¼ 7.14 Hz, -CH3), 4.06–4.13 (m, 2H, OCH2), 4.43 (t, 1H,
J ¼ 7.15 Hz, SCH), 6.56 (s, 1H, Pyr-5-H), 7.64–7.85 (m, 6H, Ph1-
2,3,5,6-H, Ph2-3,5-H), 8.00 (d, 2H, J ¼ 8.4 Hz, Ph2-2,6-H), 10.13 (s,
1H, NH), 12.95 (s, 1H, COOH). 13C-NMR (62.90 MHz, DMSO-d6) d:
13.79 (CH3-Hex), 13.93 (-CH3), 21.89 (CH2-Hex), 26.45 (CH2-Hex),
28.01 (CH2-Hex), 30.92 (CH2-Hex), 31.33 (CH2-Hex), 46.83 (SCH),
60.99 (OCH2), 101.47 (Pyr-C5), 120.87 (2C, Ph-C2 þ -C6), 126.19
(2C, Ph0-C3 þ -C5), 127.32 (2C, Ph-C3 þ -C5), 129.32 (Ph-C1),
129.95 (2C, Ph0-C2 þ -C6), 133.83 (Ph-C4), 138.75 (Ph0-C4), 143.60
(Ph-C1), 157.42 (Pyr-C4), 160.42 (Pyr-C2), 167.13 (Pyr-C6), 169.63
(COOH), 171.13 (COOH). MS (EI–) m/e: 526.6 [M – 1].
To allow the subsequent Mitsunobu reaction, the amine
group of 4-amino-2,3-xylenol has to be protected with di-tert-
butyl dicarbonate without usage of any catalysts [17]. The boc-
protected 4-amino-2,3-xylenol reacted appropriately under
Mitsunobu conditions to give 8a and 8b (Scheme 2; step
(iii)) [18]. Next, boc-protected amines 8a and 8b were depro-
tected by treatment with trifluoroacetic acid (Scheme 2; step
(iv)) [19]. Finally, hydrolysis of the ester moiety with lithium
hydroxide in a mixture of THF, water, and methanol yielded
the desired products 10a and 10b (Scheme 2; step (v)).
In summary, we have presented the successful functional-
ization of selected fatty acid mimetics. A coupleable carbox-
ylic acid group was introduced to the PA derivative 1 and a
coupleable amino group to the 2-(phenylthio)alkanoic
acid derivative 2 allowing regioselective solid-phase coupling.
We have thus provided the chemical starting point for
‘‘protein-fishing’’ experiments, which may give valuable
new insights in the pharmacology of these interesting
pharmacophores.
Experimental
40-(6-Chloro-2-(2-ethoxy-2-oxoethylthio)pyrimidin-4-
ylamino)biphenyl-4-carboxylic acid 5b [14]
General
All commercially available chemicals and solvents are of reagent
grade and were used without further purification unless speci-
0.358 g of 4b (1.35 mmol, 1 eq) and 0.337 g of 4-amino(biphenyl)
carboxylic acid hydrochloride (1.35 mmol, 1 eq) were dissolved
in 10 mL ethanol. 0.38 mL triethylamine (2.7 mmol, 2 eq) were
added and the reaction mixture was heated to 808C for 1 h. All
volatile components were removed in vacuo. The purification by
column chromatography (n-hexane/ethylacetate, 10:1; n-hexane/
ethyl acetate, 5:1; n-hexane/ethyl acetate, 3:1; ethyl acetate)
yielded in a white solid (0.57 g, 95%). 1H-NMR (300.13 MHz,
DMSO-d6) d: 1.11 (t, 3H, J ¼ 7.14 Hz, -CH3), 4.01 (s, 2H, SCH2),
4.05 (q, 2H, J ¼ 7.11 Hz, OCH2), 6.55 (s, 1H, Pyr-5-H), 7.65–7.83 (m,
6H, Ph1-2,3,5,6-H, Ph2-3,5-H), 8.00 (d, 2H, J ¼ 8.5 Hz, Ph2-2,6-H),
10.12 (s, 1H, NH), 12.92 (s, 1H, COOH). 13C-NMR (62.90 MHz,
DMSO-d6) d: 13.93 (-CH3), 32.86 (SCH2), 61.00 (OCH2), 101.31
(Pyr-C5), 120.75 (2C, Ph-C2 þ -C6), 126.19 (2C, Ph0-C3 þ -C5),
1
fied otherwise. H-NMR and 13C-NMR spectra were measured in
DMSO-d6 on a Bruker AM 250, ARX 300, and AVANCE 300 spec-
trometer (Bruker, Rheinstetten, Germany). Chemical shifts are
reported in parts per million (ppm) using tetramethylsilane
(TMS) as internal standard. Mass spectra have been performed
by the Institute of Organic Chemistry and Chemical Biology,
Goethe University of Frankfurt, on a Fisons Instruments VG
Platform II spectrometer measuring in the positive or negative
ion mode (ESI–MS system). Merck silica gel 60 (Merck, Darmstadt,
Germany) was used for column chromatography.
Chemistry
40-(6-Chloro-2-(2-ethoxy-2-oxooctan)pyrimidin-4-ylamino)
biphenyl-4-carboxylic acid 5a [14]
4-Amino(biphenyl)carboxylic acid was prepared by treatment
of 0.32 g of 4-amino(biphenyl)carboxylic acid hydrochloride
127.34
(2C,
Ph-C3 þ -C5),
129.27
(Ph-C1),
129.97
(2C, Ph0-C2 þ -C6), 133.71 (Ph-C4), 138.80 (Ph0-C4), 143.62 (Ph-C1),
157.42 (Pyr-C4), 160.42 (Pyr-C2), 167.12 (Pyr-C6), 168.60 (COOEt),
170.02 (COOH). MS (EIþ) m/e: 442.4 [M þ 1].
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Arch. Pharm. Chem. Life Sci. 2010, 10, 625–630
Functionalization of Fatty Acid Mimetics
629
1.79 (m, 2H, CH2-Bu), 1.94 (s, 3H, Ph0-3-CH3), 2.02 (s, 3H, Ph0-2-CH3),
2.05–2.17 (m, 2H, -CH2-), 3.56 (t, 1H, J ¼ 7.54 Hz, SCH), 3.92 (t, 2H,
J ¼ 6.12 Hz, Ph0-OCH2), 4.00 (q, 2H, J ¼ 7.20 Hz, OCH2), 4.13 (t,
2H, J ¼ 6.21 Hz, Ph-OCH2), 4.33 (bs, 2H, NH2), 6.42 (d, 1H,
J ¼ 8.54 Hz, Ph0-6-H), 6.55 (d, 1H, J ¼ 8.54 Hz, Ph0-5-H), 6.93 (d,
2H, J ¼ 8.72 Hz, Ph-3-H þ -5-H), 7.35 (d, 2H, J ¼ 8.72 Hz, Ph-2-
Ethyl-2-(4-(3-(4-(tert.-butoxycarbonylamino)-2,3-dimethyl-
phenoxy)propoxy)-phenylylthio) hexanoate 8a [18]
0.76 g of 7a (2.32 mmol, 1.1 eq) and 0.5 eq of (4-hydroxy-2,3-
dimethyl-phenyl)-carbamic acid tert-butyl ester (2.11 mmol, 1 eq)
were dissolved in 7 mL absolute THF. 0.61 g of PPh3 (2.32 mmol,
1.1 eq) dissolved in 3 mL absolute THF were added. Next, 0.4 g
DEAD (2.32 mmol, 1 eq) was added dropwise under cooling (ice
bath). All steps were carried out under argon atmosphere. After
stirring for 7 h at room temperature, the reaction was com-
pleted. All volatile components were removed in vacuo. The prod-
uct was purified by column chromatography with n-hexane/ethyl
acetate, 20:1; n-hexane/ethyl acetate, 15:1 and n-hexane/ethyl
13
H þ 6-H). C-NMR (75.45 MHz, DMSO-d6) d: 12.01 (Ph0-2-CH3),
13.24 (Ph0-3-CH3), 13.70 (CH3-Bu), 13.88 (-CH3), 21.68 (CH2-Bu),
28.65 (CH2-Bu), 28.89 (-CH2-), 30.48 (CH2-Bu), 50.45 (SCH), 60.38
(-OCH2), 64.59 (Ph-OCH2), 65.36 (Ph0-OCH2), 111.15 (Ph0-C6), 112.05
(Ph0-C5), 115.05 (2C, Ph-C3 þ -C5), 121.43 (Ph0-C4), 122.40 (Ph-C1),
124.91 (Ph0-C2), 135.82 (2C, Ph-C2 þ -C6), 140.42 (Ph0-C3), 148.16
(Ph0-C1), 159.02 (Ph-C4), 171.46 (COO). MS (EIþ) m/e: 446.7 [M þ 1].
acetate) The product is
a
clear oil (1.0 g, 79%). 1H-NMR
(300.13 MHz, DMSO-d6) d: 0.83 (t, 3H, J ¼ 6.68 Hz, CH3-Bu), 1.06
(t, 3H, J ¼ 7.07 Hz, -CH3), 1.19–1.36 (m, 4H, CH2-Bu), 1.41 (s,
9H, CH3-t-Bu), 1.48–1.77 (m, 2H, CH2-Bu), 2.02 (s, 3H, Ph0-3-CH3),
2.06 (s, 3H, Ph0-2-CH3), 2.16 (t, 2H, J ¼ 6.17 Hz, -CH2-), 3.56 (t, 1H,
J ¼ 7.59 Hz, SCH), 3.99 (q, 2H, J ¼ 7.21 Hz, OCH2), 4.06 (t, 2H,
J ¼ 6.19 Hz, Ph0-OCH2), 4.15 (t, 2H, J ¼ 6.19 Hz, Ph-OCH2), 6.74 (d,
1H, J ¼ 8.89 Hz, Ph0-6-H), 6.93 (d, 2H, Ph-3-H þ -5-H þ 1H, Ph0-5-H),
7.35 (d, 2H, J ¼ 8.69 Hz, Ph-2-H þ -6-H), 8.37 (bs, 1H, NH). 13C-NMR
(75.44 MHz, DMSO-d6) d: 11.99 (Ph0-2-CH3), 13.68 (CH3-Bu), 13.87
(-CH3), 14.41 (Ph0-3-CH3), 21.66 (CH2-Bu), 28.15 (3C, CH3-t-Bu), 28.64
(2C, -CH2- þ CH2-Bu), 30.50 (CH2-Bu), 50.49 (SCH), 60.37 (-OCH2),
64.60 (2C, Ph-OCH2 þ Ph0-OCH2), 78.08 (C(CH3)3-t-Bu), 109.03 (Ph0-
C6), 115.09 (2C, Ph-C3 þ -C5), 122.52 (Ph-C1), 124.28 (Ph0-C5), 124.57
(Ph0-C2), 129.50 (Ph0-C4), 133.34 (Ph0-C3), 135.78 (2C, Ph-C2 þ -C6),
153.82 (Ph0-C1), 154.16 (COO-t-Bu), 158.97 (Ph-C4), 171.45 (COO). MS
(EIþ) m/e: 546.9 [M þ 1].
Ethyl-2-(4-(3-(4-amino-2,3-dimethylphenoxy)propoxy)-
phenylylthio) acetate 9b [19]
1H-NMR (300.13 MHz, DMSO-d6) d: 1.10 (t, 3H, J ¼ 7.09 Hz, -CH3),
1.94 (s, 3H, Ph0-3-CH3), 2.02 (s, 3H, Ph0-2-CH3), 2.04–2.15 (m, 2H, -
CH2-), 3.67 (s, 2H, SCH2), 3.92 (t, 2H, J ¼ 6.11 Hz, Ph0-OCH2), 4.03
(q, 2H, J ¼ 7.14 Hz, OCH2), 4.12 (t, 2H, J ¼ 6.22 Hz, Ph-OCH2), 4.32
(bs, 2H, NH2), 6.42 (d, 1H, J ¼ 8.64 Hz, Ph0-6-H), 6.54 (d, 1H,
J ¼ 8.64 Hz, Ph0-5-H), 6.92 (dd, 2H, J1 ¼ 2.22 Hz, J2 ¼ 6.78 Hz,
Ph-3-H þ -5-H), 7.34 (dd, 2H, J1 ¼ 2.22 Hz, J2 ¼ 6.76 Hz, Ph-2-
13
H þ -6-H). C-NMR (75.45 MHz, DMSO-d6) d: 12.02 (Ph0-2-CH3),
13.25 (-CH3), 13.92 (Ph0-3-CH3), 28.91 (-CH2-), 36.96 (SCH2), 60.66
(-OCH2), 64.57 (Ph-OCH2), 65.37 (Ph0-OCH2), 111.14 (Ph0-C6), 112.05
(Ph0-C5), 115.19 (2C, Ph-C3 þ -C5), 121.43 (Ph0-C4), 124.69 (Ph-C1),
124.91 (Ph0-C2), 132.78 (2C, Ph-C2 þ -C6), 140.42 (Ph0-C3), 148.17
(Ph0-C1), 158.16 (Ph-C4), 169.37 (COO). MS (EIþ) m/e: 390.3 [M þ 1].
Ethyl 2-(4-(3-(4-(tert-butoxycarbonylamino)-2,3-dimethyl-
2-(4-(3-(4-Amino-2,3-dimethylphenoxy)propoxy)
phenylylthio)-hexanoic acid 10a
phenoxy)propoxy)-phenylylthio) acetate 8b [18]
1H-NMR (250.13 MHz, DMSO-d6) d: 1.09 (t, 3H, J ¼ 7.1 Hz, -CH3),
1.41 (s, 9H, CH3-t-Bu), 2.02 (s, 3H, Ph0-3-CH3), 2.06 (s, 3H, Ph0-2-CH3),
2.15 (t, 2H, J ¼ 6.09 Hz, -CH2-), 3.66 (s, 2H, SCH2), 3.97–4.09 (t, 2H,
Ph-OCH2 þ q, 2H, OCH2), 4.13 (t, 2H, J ¼ 6.10 Hz, Ph0-OCH2), 6.74
(d, 1H, J ¼ 8.75 Hz, Ph0-6-H), 6.92 (d, 2H, Ph-3-H þ -5-H þ 1H, Ph0-
5-H), 7.34 (d, 2H, J ¼ 8.73 Hz, Ph-2-H þ -6-H), 8.36 (bs, 1H, NH). 13C-
NMR (75.44 MHz, DMSO-d6) d: 11.99 (Ph0-2-CH3), 13.91 (-CH3), 14.43
(Ph0-3-CH3), 28.15 (3C, CH3-t-Bu), 28.67 (-CH2-), 36.96 (SCH2), 60.66 (-
OCH2), 64.48 (Ph0-OCH2), 64.56 (Ph-OCH2), 78.09 (C(CH3)3-t-Bu),
108.98 (Ph0-C6), 115.20 (2C, Ph-C3), 124.29 (Ph0-C5), 124.54 (Ph0-
C2), 124.74 (Ph-C1), 129.46 (Ph0-C4), 132.78 (2C, Ph-C2 þ -C6),
133.35 (Ph0-C3), 153.81 (Ph0-C1), 154.17 (COO-t-Bu), 158.12 (Ph-C4),
169.37 (COO). MS (EIþ) m/e: 490.4 [M þ 1].
0.22 g of 9a (0.61 mmol, 1 eq) was dissolved in 12.4 mL THF and
5 eq) in
4.1 mL methanol. 72.8 mg of LiOH (3.04 mmol,
4.1 mL H2O were added at 08C. The reaction mixture was stirred
at 40 to 508C for 1.5 h. Subsequently, the solvent was removed
in vacuo and the remaining residue was dissolved in H2O. The
aqueous phase was neutralized with 2 M HCl and extracted with
ethyl acetate. The organic layer was dried over MgSO4 and con-
centrated in vacuo. Finally, the crude product was recrystallized
in methanol to give purified 10a (0.135 g, 53%). 1H-NMR
(300.13 MHz, DMSO-d6) d: 0.82 (t, 3H, J ¼ 6.93 Hz, CH3-Bu),
1.15–1.45 (m, 4H, CH2-Bu), 1.45–1.79 (m, 2H, CH2-Bu), 1.93 (s,
3H, Ph0-3-CH3), 2.02 (s, 3H, Ph0-2-CH3), 2.04–2.16 (m, 2H, -CH2-),
3.48 (t, 1H, J ¼ 6.26 Hz, SCH), 3.92 (t, 2H, J ¼ 6.15 Hz, Ph0-OCH2),
4.12 (t, 2H, J ¼ 6.15 Hz, Ph-OCH2), 6.42 (d, 1H, J ¼ 8.65 Hz, Ph0-6-
H), 6.55 (d, 1H, J ¼ 8.65 Hz, Ph0-5-H), 6.92 (d, 2H, J ¼ 8.63 Hz, Ph-3-
H þ -5-H), 7.35 (d, 2H, J ¼ 8.63 Hz, Ph-2-H þ -6-H). 13C-NMR
(75.45 MHz, DMSO-d6) d: 12.03 (Ph0-2-CH3), 13.25 (Ph0-3-CH3),
13.72 (CH3-Bu), 21.73 (CH2-Bu), 28.71 (CH2-Bu), 28.96 (-CH2-),
30.88 (CH2-Bu), 50.97 (SCH), 64.62 (Ph-OCH2), 65.45 (Ph0-OCH2),
111.19 (Ph0-C6), 112.13 (Ph0-C5), 115.06 (2C, Ph-C3 þ -C5), 121.50
(Ph0-C4), 123.38 (Ph-C1), 124.96 (Ph0-C2), 135.08 (2C, Ph-C2 þ -C6),
140.38 (Ph0-C3), 148.26 (Ph0-C1), 158.71 (Ph-C4), 173.00 (COO). MS
(EIþ) m/e: 418.3 [M þ 1].
Ethyl-2-(4-(3-(4-amino-2,3-dimethylphenoxy)propoxy)-
phenylylthio) hexanoate 9a [19]
0.47 g of 8a (0.86 mmol, 1 eq) were dissolved in CH2Cl2. The
reaction mixture was cooled to 08C (ice bath) and 1.92 mL TFA
(25.9 mmol, 30 eq) were added. After removal of the ice bath and
stirring at room temperature for 45 min, the reaction was com-
pleted. All volatile components were removed in vacuo. The res-
idue was co-evaporated with toluene, then with methanol, and
finally dissolved in ethyl acetate. The organic layer was extracted
with 2 M HCl, saturated NaHCO3 solution and brine. The remain-
ing organic phase was dried over MgSO4 and concentrated
in vacuo. The product is an orange oil (0.28 g, 73%). 1H-NMR
(300.13 MHz, DMSO-d6) d: 0.83 (t, 3H, J ¼ 6.68 Hz, CH3-Bu),
1.07 (t, 3H, J ¼ 6.99 Hz, -CH3), 1.15–1.45 (m, 4H, CH2-Bu), 1.46–
2-(4-(3-(4-Amino-2,3-dimethylphenoxy)propoxy)
phenylylthio)-acetic acid 10b
1H-NMR (300.13 MHz, DMSO-d6) d: 1.94 (s, 3H, Ph0-3-CH3), 2.02 (s,
3H, Ph0-2-CH3), 2.06–2.15 (m, 2H, -CH2-), 3.61 (s, 2H, SCH2), 3.92 (t,
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

630
M. Dittrich et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 625–630
2H, J ¼ 5.65 Hz, Ph0-OCH2), 4.11 (t, 2H, J ¼ 5.65 Hz, Ph-OCH2),
6.42 (d, 1H, J ¼ 8.47 Hz, Ph0-6-H), 6.55 (d, 1H, J ¼ 8.47 Hz, Ph0-5-H),
6.91 (d, 2H, J ¼ 8.39 Hz, Ph-3-H þ -5-H), 7.32 (d, 2H, J ¼ 8.31 Hz,
[7] H. Zettl, R. Steri, M. La¨mmerhofer, M. Schubert-Zsilavecz,
Bioorg. Med. Chem. Lett. 2009, 19, 4421–4426.
[8] M. W. Harding, A. Galat, D. E. Uehling, S. L. Schreiber, Nature
1989, 341, 758–760.
13
Ph-2-H þ -6-H). C-NMR (75.45 MHz, DMSO-d6) d: 12.09 (Ph0-2-
CH3), 13.31 (Ph0-3-CH3), 28.97 (-CH2-), 37.19 (SCH2), 64.59 (Ph-
OCH2), 65.40 (Ph0-OCH2), 111.14 (Ph0-C6), 112.14 (Ph0-C5), 115.22
(2C, Ph-C3 þ -C5), 121.55 (Ph0-C4), 124.94 (Ph-C1), 125.49 (Ph0-C2),
131.99 (2C, Ph-C2 þ -C6), 140.38 (Ph0-C3), 148.24 (Ph0-C1), 157.86
(Ph-C4), 170.78 (COO). MS (EIþ) m/e: 362.1 [M þ 1].
[9] J. Taunton, C. A. Hassig, S. L. Schreiber, Science 1996, 272,
408–411.
[10] L. Tausch, A. Henkel, U. Siemoneit, D. Poeckel, et al.,
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The authors have declared no conflict of interest.
[12] K. Amatschek, J. High Resolut. Chromatogr. 2000, 23,
47–58.
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ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com