Design and Synthesis of Highly Active Peroxisome Proliferator-Activated Receptor (PPAR) β/δ Inverse Agonists with Prolonged Cellular Activity

Article abstract of DOI:10.1002/cmdc.201500594

Based on 3-(((4-(hexylamino)-2-methoxyphenyl)amino)sulfonyl)-2-thiophenecarboxylic acid methyl ester (ST247, compound 2), a recently described peroxisome proliferator-activated receptor (PPAR)β/δ-selective inverse agonist, we designed and synthesized a se

Full text of DOI:10.1002/cmdc.201500594

DOI: 10.1002/cmdc.201500594
Full Papers
Design and Synthesis of Highly Active Peroxisome
Proliferator-Activated Receptor (PPAR) b/d Inverse
Agonists with Prolonged Cellular Activity
Philipp M. Toth,[a] Sonja Lieber,[b] Frithjof M. Scheer,[a] Tim Schumann,[b] Yvonne Schober,[c]
Wolfgang A. Nockher,[c, d] Till Adhikary,[b] Sabine Müller-Brüsselbach,[b] Rolf Müller,[b] and
Wibke E. Diederich*[a, e]
Based on 3-(((4-(hexylamino)-2-methoxyphenyl)amino)sulfonyl)-
2-thiophenecarboxylic acid methyl ester (ST247, compound 2),
a recently described peroxisome proliferator-activated receptor
(PPAR)b/d-selective inverse agonist, we designed and synthe-
sized a series of structurally related ligands. The structural
modifications presented herein ultimately resulted in a series
of ligands that display increased cellular activity relative to 2.
Moreover, with methyl 3-(N-(2-(2-ethoxyethoxy)-4-(hexylami-
no)phenyl)sulfamoyl)thiophene-2-carboxylate (PT-S264, com-
pound 9u), biologically relevant plasma concentrations in mice
were achieved. The compounds presented in this study will
provide useful novel tools for future investigations addressing
the role of PPARb/d in physiological and pathophysiological
processes.
Introduction
The three peroxisome proliferator-activated receptor (PPAR)
subtypes a (NR1C1), b/d (NR1C2), and g (NR1C3) are members
of the nuclear receptor family, a group of transcription factors
regulated by endogenous fatty acids or synthetic ligands.[1] De-
pending on the nature of the interacting ligand, they modulate
the transcription of their target genes by recruiting either co-
activators (agonists) or co-repressors (inverse agonists), thus in-
ducing or repressing transcription.
Although several PPAR ligands are currently in clinical use
(e.g., lipid-lowering fibrates for PPARa and antidiabetic glita-
zones for PPARg), no drug has been approved for PPARb/d.
PPARb/d is expressed in many cell types and hence involved in
diverse biological processes, such as lipid and glucose metabo-
lism,[2] cell differentiation,[3] proliferation and apoptosis,[4,5]
immune regulation,[6] and tumorigenesis,[7,8] thus representing
an attractive pharmacological target. While its role in the lipid
and glucose metabolism is well documented, and an agonist
had entered clinical trials,[9] its role in cell differentiation, prolif-
eration, and tumorigenesis is not yet fully understood.[5,7,8,10]
We recently demonstrated that inverse PPARb/d agonists re-
press the ANGPTL4-mediated invasion of human breast cancer
cells into a three-dimensional matrix in vitro, indicating that se-
lectively repressing PPARb/d target genes could be advanta-
geous under certain pathophysiological conditions.[11]
[a] Dr. P. M. Toth, Dr. F. M. Scheer, Prof. Dr. W. E. Diederich
Institut für Pharmazeutische Chemie
Zentrum für Tumor- und Immunbiologie (ZTI)
Philipps-Universität Marburg
Hans-Meerwein-Straße 3, 35043 Marburg (Germany)
E-mail: wibke.diederich@staff.uni-marburg.de
[b] Dr. S. Lieber, T. Schumann, Dr. T. Adhikary, Dr. S. Müller-Brüsselbach,
Prof. Dr. R. Müller
The development of subtype-selective and bioavailable li-
gands with a distinct profile (agonist, inverse agonist, antago-
nist) is a prerequisite to elucidate the complex system of
PPARb/d-mediated transcriptional processes, especially in tu-
morigenesis in vivo. This may not only result in a better under-
standing of the underlying processes, but also clarify whether
PPARb/d is a valid target for the development of new chemo-
therapeutics. However, as research has so far predominantly fo-
cused on the activation of PPARb/d rather than on its inhibi-
tion, the detailed exploration of PPARb/d functions is mainly
hampered by the lack of subtype-selective, bioavailable and
highly potent inverse agonistic or antagonistic ligands, where-
as agonistic ligands are abundant.[1,12]
Institut für Molekularbiologie und Tumorforschung (IMT)
Zentrum für Tumor- und Immunbiologie (ZTI)
Philipps-Universität Marburg
Hans-Meerwein-Straße 3, 35043 Marburg (Germany)
[c] Dr. Y. Schober, Dr. W. A. Nockher
Institut für Laboratoriumsmedizin und Pathobiochemie
Molekulare Diagnostik
Philipps-Universität Marburg, Baldingerstraße, 35043 Marburg (Germany)
[d] Dr. W. A. Nockher
Core Facility Metabolomics
Philipps-Universität Marburg, Baldingerstraße, 35043 Marburg (Germany)
[e] Prof. Dr. W. E. Diederich
Core Facility Medicinal Chemistry
Philipps-Universität Marburg, Hans-Meerwein-Straße 3, 35043 Marburg
(Germany)
GSK0660 (1), disclosed in 2008, was the first PPARb/d-selec-
tive inverse agonist described, but suffered from rapid clear-
ance in rodents (Figure 1).[13] Development of stilbene deriva-
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201500594.
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Figure 1. Structures of GSK0660 (1),[13] ST247 (2),[16,17] PT-S58 (3),[16,17] and
DG172 (4).[14]
tive DG172 (4),[14] resulted in an orally available PPARb/d-selec-
tive inverse agonist, but turned out to exhibit off-target effects
in myeloid cells.[15] Based on the structure of 1, we previously
reported a structure–activity relationship (SAR) study which led
to the identification of ST247 (2), being one of the most active
inverse PPARb/d agonists known to date. In the same study,
we also described PT-S58 (3), representing the first pure
PPARb/d antagonist.[11,16,17]
However, it turned out that 2 was seemingly unsuitable for
long-term cellular experiments aimed to elucidate the effects
of PPARb/d inhibition, especially in cell differentiation. More-
over, with 2 and related analogues, no relevant plasma con-
centrations could be reached after oral and even intravenous
application. We therefore revisited our initial SAR, which had
led to the identification of 2 focusing on the further improve-
ment of this ligand class.
Figure 2. PPARb/d binding site (blue mesh) with EPA bound in two different
conformations (yellow and red spheres), PDB ID: 3GWX;[18] image created
using PyMOL 0.99.[19]
Chemistry
All subsequent compounds were synthesized using a four-step
procedure starting from commercially available 2-nitro-5-fluo-
rophenol (5), which was alkylated with various alkyl halides
(Scheme 1). These reactions proceeded smoothly, giving rise to
the corresponding aryl alkyl ethers, in most cases without the
need for further purification by column chromatography, with
yields ranging from 40 to 95%. For cases in which an alkyl
chloride or bromide was used, a catalytic amount of sodium
iodide was added to facilitate the reaction. The following syn-
thetic steps of this sequence were carried out as described
Results and Discussion
The large ligand binding pocket of PPARb/d has a volume of
~1300 Š3, is Y-shaped and, except for a small polar section,
mainly lipophilic. Flexible ligands such as the fatty acid eicosa-
pentaenoic acid (EPA) adopt different conformations within the
binding pocket, varying only in the occupation of the hydro-
phobic sub-pockets (Figure 2).[18]
Based on these findings we envisioned that by introducing
an additional substituent into 2 we would not only be able to
better mimic the Y shape of the binding cleft, but also increase
hydrophobic interactions within the binding pocket, which
should be advantageous for the binding affinity of the ligands.
As revealed by our initial SAR studies, a significant decrease in
binding affinity to the PPARb/d ligand binding domain (LBD) is
observed if the methoxy substituent of 1 is moved from its
original position adjacent to the sulfonamide or if it is com-
pletely missing. Interestingly though, replacement of the me-
thoxy moiety by a hydrogen substituent in 2 resulted in a com-
pound being less potent than 2 but still significantly more
potent than the initial lead 1. Further screening of various sub-
stitution patterns at the central phenyl moiety of 2 (data not
shown) finally confirmed that the 2-position seems to be opti-
mal for further structural variations (Figure 2).
Scheme 1. Synthesis of PPARb/d inverse agonists. Reagents and conditions:
a) R1I, K2CO3, acetone, 708C; b) R1Cl/Br, NaI, acetone, 708C; c) R2NH2, MeCN,
1208C, microwave irradiation; d) Pd/C, H2, EtOAc, RT; e) methyl 3-(chlorosul-
fonyl)thiophene-2-carboxylate, DMAP, pyridine, RT.
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Table 1. PPARb/d inverse agonists of the different series.
Compd
R1
R2
Compd
R1
R2
Compd
R1
R2
9a
nHex
9h
nHex
9o
Me
9b
9c
nHex
nHex
9i
9j
nHex
nHex
9p
9q
Me
Me
9d
nHex
9k
nHex
9r
Me
Me
9e
9 f
nHex
nHex
9l
Me
Me
9s
9t
9m
nHex
nHex
9g
nHex
9n
Me
9u
previously,[16] rendering the sulfonamides 9a–w in overall
yields ranging from 8 to 53% over four steps (Scheme 1 and
Table 1).
pounds with respect to assay conditions. Besides the imple-
mentation of non-ionizable functionalities such as polyhydroxy
or polyethylene glycol moieties, the introduction of polar, pro-
tonatable functionalities is a valid and frequently applied
methodology.[20]
Variation of the methoxy and amino substituents
Consequently, we introduced an ethoxypropyl moiety as
well as a variety of more polar alkyl amine residues at R2 (9l–
9s; Table 1 and Table S2). Most modifications of the n-hexyl
moiety resulted in a significant decrease in the ability to recruit
co-repressor peptides, as revealed in the TR-FRET assay.
Only the morpholinopropyl-substituted derivative 9s clearly
exceeded the inverse-agonistic effect observed for 2 (Table 1
and Table S2). Thus, in a third series the n-pentyl substituent at
position R1 (9d) was replaced by the equally sized but signifi-
cantly more polar methoxypropoxy and 2-ethoxyethoxy resi-
dues, respectively (9t, 9u; Table 1 and Table S3). Because the
ethoxyethoxy-substituted derivative 9u (PT-S264) was signifi-
cantly more potent than 2 in the TR-FRET assay, albeit less
active than 9d, we considered further testing of 9u (PT-S264)
to be worthwhile (Figure 3 and Table S3).
As the initial SAR had revealed that the effect of the different
substituents at the 4’-amino position (R2) and the central 1,4-
diaminobenzene moiety (R1) on the ligand’s affinity toward the
PPARb/d LBD is merely additive, we focused first on evaluating
the optimal size and shape of the attached side chains R1 and
R2 by introducing linear and branched alkyl chains as well as
alkyl-chain-linked aryl moieties and several amines (Scheme 1
and Table 1). The synthesized compounds were tested in
a qualitative manner for their inverse-agonistic effect by meas-
uring the recruitment of co-repressor peptides in a time-
resolved fluorescence resonance energy transfer (TR-FRET)
assay in vitro in comparison with 1 and 2 (Tables S1–S3, Sup-
porting Information).
In an initial series, we focused on evaluating the optimal size
and shape of the attached side chain R1 by introducing linear
and branched alkyl chains as well as alkyl-chain-linked aryl
moieties (9a–9k). Interestingly, the introduction of linear alkyl
chains increases the recruitment of co-repressors significantly
for some of the compounds, with 9d (Table 1 and Table S1)
having the strongest effect, albeit the underlying SAR is not
linear. However, as 9d, being equipped with an additional hy-
drophobic moiety, was even more lipophilic than 2, we decid-
ed to focus next on optimizing the solubility of the com-
In the final series, we combined the substitution patterns of
the most active derivatives of both series and synthesized the
corresponding ligands 9v and 9w bearing a morpholinopro-
pylamino substituent as well as a pentyl or ethyloxyethyl sub-
stituent (Table 2). To elucidate their potency further, 9d and
9u–w as well as 2 as reference were tested with respect to
their ability to repress the PPARb/d target genes Angptl4, PDK4,
and Cpt1a in cell-based assays. As illustrated in Figure 3, the
newly designed ligands showed a 2.5- to 5-fold enhanced re-
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Table 2. Inhibitory activity of selected PPARb/d inverse agonists.
Compd
R1
R2
IC50 [nm][a]
Angptl4
PDK4
16.17
3.03
2
Me
nHex
nHex
3.84
2.42
9d
9u
9v
nHex
6.43
4.56
9.08
11.12
9w
52.22
46.96
[a] Relative expression.
In contrast to 2 and 1, for 9u, biologically relevant plasma con-
centrations could be determined 120 min post-application.
Thus 9u represents the first member of this ligand class that
might prove useful for further in vivo experiments.
Conclusions
Systematic structural optimization of the PPARb/d-inverse ago-
nist 2 resulted in a series of ligands displaying significantly in-
creased potency relative to 2. Compounds 9d and 9u (PT-
S264) exhibited prolonged activity in cellular assays. Moreover,
one of the ligands (9u) gave rise to biologically relevant
plasma concentrations in mice. Compound 9u (PT-S264) will
therefore not only be a useful tool to address the role of
PPARb/d in physiological and pathophysiological processes in
vitro but also in vivo. The extended half-life of the compound
in cell-based assays will enable studies which require several
days of cultivation, such as differentiation processes.
Figure 3. Concentration-dependent repression of the a) Angptl4 gene in
C2C12 cells and the b) PDK4 gene in MDA-MB-231 cells by derivatives of
parent compound 2. Cells were treated with the indicated concentrations
for 24 h and analyzed by RT-qPCR. c) Repression of the Cpt1a gene in C2C12
cells by derivatives of the parent compound 2. Cells were treated with the
indicated compounds at a concentration of 1 mm for 24 h and analyzed by
RT-qPCR. DMSO: solvent control. Values are the average ÆSD of triplicates;
significant difference from DMSO-treated sample: ***p<0.001, **p<0.01,
and *p<0.05 (t-test).
Experimental Section
Chemistry
Materials and methods. Unless stated otherwise, all commercially
available starting materials and solvents were purchased and used
without further purification. Thin-layer chromatography (TLC) was
performed on pre-coated TLC plates (silica gel 60 F254 Merck). Flash
column chromatography was performed on prepacked flash chro-
matography columns (PF 30-SIHP-JP/12G) purchased from Inter-
chim using a Büchi separation system. Cyclohexane p.a. (from
Grüssing), EtOAc p.a. (Sigma–Aldrich or Acros), were distilled prior
to use. HPLC-quality acetonitrile was obtained from VWR. Pyridine
was kept over KOH for 48 h, distilled, and stored over molecular
pression of the respective genes relative to the hitherto most
active compound 2.
Besides IC50 and subtype specificity (Figure S1) determina-
tion for the two derivatives 9d and 9u, their impact on PDK4
expression in primary human monocyte-derived macrophages
was evaluated over a time period of six days (Figure 4). The ex-
perimental data clearly reveal the superior effect of both com-
pounds relative to 2. There is no discernible loss of transcrip-
tional repression within the observation period in cells treated
with 9d or 9u (PT-S264), while PDK4 transcription rises steadily
in 2-treated cells after its initial repression.
1
sieves (4 Š) under an argon atmosphere. H and 13C NMR spectra
were recorded on Jeol ECX-400 or Jeol ECA-500 spectrometers.
Chemical shifts (d) are given in ppm with the residual solvent
signal used as reference (CDCl3: s, 7.26 ppm (1H) and t, 77.1 ppm
(13C); [D6]DMSO: quint, 2.50 ppm (1H) and quint, 40.1 ppm (13C)).
Spectra with CDCl3 as solvent were recorded at ambient tempera-
Finally, 9u was administered i.v. to mice, and plasma con-
centrations were determined at various time points (Figure 5).
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Figure 4. Kinetics of PDK4 repression by inverse agonists in primary human monocyte-derived macrophages. After six days of differentiation, cells were treat-
ed with the compounds (300 nm) and harvested at the indicated time points. RT-qPCR was performed. Expression values were calculated relative to solvent
control (DMSO-treated cells). Error bars represent standard deviations derived from technical triplicates. A representative experiment is shown. Significant dif-
ference from DMSO-treated sample: ***p<0.001, **p<0.01, and *p<0.05 (t-test).
under reduced pressure to give the pure products without further
purification.
General procedure 2 for the synthesis of N-alkyl-4-nitro-3-alkox-
yanilines (7a–w): Procedure A: The corresponding 2-alkoxy-4-
fluoro-1-nitrobenzene (6a–m, 1.0 equiv) and the respective amine
(4.0 equiv) were dissolved in acetonitrile (2–4 mL) while stirring.
The reaction mixture was heated at 1208C by microwave irradia-
tion in a sealed 10 mL reaction vessel for 60 min. Subsequent re-
moval of the solvent and purification by flash column chromatog-
raphy (cyclohexane/EtOAc or dichloromethane/MeOH) yielded the
target compounds. Procedure B: The corresponding 2-alkoxy-4-
fluoro-1-nitrobenzene (6a–m, 1.0 equiv), the respective amine (1.0–
2.0 equiv) and triethylamine (3.0–5.0 equiv) were dissolved in ace-
tonitrile (2–4 mL) while stirring. The reaction mixture was heated at
1208C by microwave irradiation in a sealed reaction vessel for
Figure 5. Plasma concentration of 9u after administration (1 mgkgÀ1 i.v.) in
60 min. Subsequent removal of solvent and purification by flash
column chromatography (cyclohexane/EtOAc or dichloromethane/
MeOH) yielded the target compounds.
mice. Values are the average ÆSD of n=3 biological replicates.
ture while spectra with [D6]DMSO as solvent were recorded at
General procedure 3 for the synthesis of 1,4-phenylenediamines
30.08C. Coupling constants (J) are reported in Hz. Mass spectra
(8q–w): The corresponding N-alkyl-4-nitro-3-alkoxyanilines (7a–w,
were recorded on a double-focusing sector field spectrometer type
1.0 equiv) were dissolved in EtOAc (~10 mLmmolÀ1) and Pd/C
70/70 H (Vacuum Generators), on a double-focusing sector field
(10% w/w) was added (26 mgmmolÀ1). After air removal and flush-
spectrometer type AutoSpec (Micromass), on a triple quadrupole
ing with hydrogen the reaction mixture was stirred at RT under
spectrometer type EP 10+ (MS Vision) or on a triple quadrupole
light protection for 24 h. Filtration through a pad of Celite and
spectrometer type Q-Trap 2000 (Applied Biosystems). Elemental
concentration in vacuo gave the light-sensitive amines, which were
combustion analyses were performed on a Vario MICRO cube (Ele-
directly used in the next step without further purification.
mentarAnalysensysteme GmbH). Microwave irradiation was applied
General procedure 4 for the synthesis of sulfonamides (9a–w):
The corresponding 1,4-phenylenediamines (8a–w, 1.0 equiv),
DMAP (0.5 equiv) and methyl 3-(chlorosulfonyl)thiophene-2-carbox-
ylate (1.1 equiv) were dissolved in anhydrous pyridine
(10 mLmmolÀ1) while stirring under an argon atmosphere. The re-
action mixture was stirred for 48 h protected from light. Subse-
quent removal of the solvent and purification by flash column
chromatography (cyclohexane/EtOAc or dichloromethane/MeOH)
yielded the target compounds.
with a Discover BenchMate Plus (CEM GmbH). Melting points were
determined using a melting point meter KSP1N (A. KrüssOptronic
GmbH) and are uncorrected. Unless stated otherwise, all tested
compounds were at least 95% pure as determined by combustion
analysis. All compounds were synthesized following a synthetic
procedure as described previously.[16] However, for the synthesis of
branched ligands an additional synthetic step for the generation of
2-alkoxy-4-fluoro-1-nitrobenzenes was introduced.
General procedure 1 for the synthesis of 2-alkoxy-4-fluoro-1-ni-
trobenzenes (6a–m): 5-fluoro-2-nitrophenol (5, 1.0 equiv) and the
respective alkyl halide (1.5 equiv) were dissolved in dry acetone
(10 mL). Potassium carbonate (2.0 equiv) and, if a chloride or bro-
mide was used, potassium iodide (0.5 equiv) were added. The reac-
tion mixture was then heated at reflux until complete conversion
of the starting material occurred (TLC). The solvent was subse-
quently removed under reduced pressure and the resulting residue
was suspended in dichloromethane. After the addition of sodium
sulfate, the mixture was filtered and the solvent was removed
4-Fluoro-1-nitro-2-(pentyloxy)ben-
zene (6d): Compound 6d was pre-
pared according to general proce-
dure 1 using 5 (323 mg, 2.06 mmol)
and
1-iodopentane
(593 mg,
3.00 mmol, 1.5 equiv). The title com-
pound was obtained as a yellow liquid (408 mg, 87%): 1H NMR
3
4
(400 MHz, CDCl3): d=7.93 (dd, JH,H =9.0 Hz, JH,F =6.1 Hz, 1H, 6-H),
3
6.76 (dd, 3JH,F =10.5 Hz, 4JH,H =2.5 Hz, 1H, 3-H), 6.70 (ddd, JH,F
=
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9.1 Hz, 3JH,H =7.4 Hz, 4JH,H =2.5 Hz, 1H, 5-H), 4.07 (t, 3JH,H =6.4 Hz,
2H, 1’-H2), 1.89–1.82 (m, 2H, 2’-H2), 1.51–1.44 (m, 2H, 3’-H2), 1.43–
3
1.34 (m, 2H, 4’-H2), 0.94 ppm (t, JH,H =7.2 Hz, 3H, 5’-H3); 13C NMR
(100 MHz, CDCl3): d=165.8 (d, 1JC,F =255.3 Hz, 1C, C-4), 154.9 (d,
3JC,F =11.6 Hz, 1C, C-2), 136.2 (d, 4JC,F =2.9 Hz, 1C, C-1), 128.1 (d,
3JC,F =11.6 Hz, 1C, C-6), 107.1 (d, 2JC,F =23.1 Hz, 1C, C-5), 102.1 (d,
2JC,F =26.0 Hz, 1C, C-3), 70.2 (1C, C-1’), 28.5 (1C, C-2’), 28.0 (1C, C-3’),
22.4 (1C, C-4’), 14.0 ppm (1C, C-5’); MS (EI): m/z (%)=141 (57)
[MÀC5H10O]+, 157 (93) [MÀC5H10]+, 227 (100) [M]+; HRMS (EI) m/z
[M]+: calcd for C11H14FNO3: 227.095772, found: 227.097703: Anal.
calcd for C11H14FNO3: C 58.14%, H 6.21%, N 6.16%; found: C
58.59%, H 6.30%, N 6.34%.
2.0 equiv) with triethylamine (202 mg, 2.00 mmol, 2.0 equiv) as ad-
ditional base. The title compound was obtained as a yellow solid
(297 mg, 96%): mp: 63.8–65.58C; 1H NMR (400 MHz, CDCl3): d=
3
3
4
7.95 (d, JH,H =8.9 Hz, 1H, 5-H), 6.13 (dd, JH,H =9.2 Hz, JH,H =2.1 Hz,
4
1H, 6-H), 6.10 (d, JH,H =2.1 Hz, 1H, 2-H), 4.39 (brs, 1H, NH), 4.20 (t,
3JH,H =5.0 Hz, 2H, 1’’-H2), 3.87 (t, 3JH,H =5.0 Hz, 2H, 2’’-H2), 3.65 (q,
3JH,H =7.0 Hz, 2H, 4’’-H2), 3.16 (t, 3JH,H =7.2 Hz, 2H, 1’-H2), 1.64 (tt,
3
3JH,H =7.3 Hz, JH,H =7.3 Hz, 2H, 2’-H2), 1.44–1.29 (m, 6H, 3’-H2, 4’-H2,
2-(2-Ethoxyethoxy)-4-fluoro-1-nitrobenzene (6m): Compound
6m was prepared following a slightly altered general procedure 1
with a higher amount of potassium
3
3
5’-H2), 1.23 (t, JH,H =7.0 Hz, 3H, 5’’-H3), 0.90 ppm (brt, JH,H =6.8 Hz,
3H, 6’-H3); 13C NMR (126 MHz, CDCl3): d=156.4 (1C, C-3), 154.1 (1C,
C-1), 129.2 (1C, C-5), 129.1 (1C, C-4), 104.6 (1C, C-6), 96.2 (1C, C-2),
69.4 (1C, C-2’’), 68.6 (1C, C-1’’), 67.2 (1C, C-4’’), 43.5 (1C, C-1’), 31.5
(1C, C-2’), 29.1 (1C, C-4’), 26.7 (1C, C-3’), 22.6 (1C, C-5’), 15.3 (1C, C-
5’’), 14.1 ppm (1C, C-6’); MS (ESI): m/z (%)=193 (15), 265 (100)
[MÀC2H5O]+, 311 (93) [M+H]+, 333 (61) [M+Na]+; HRMS (ESI) m/z
[M+H]+ calcd for C16H27N2O4: 311.197083, found: 311.194174;
Anal. calcd for C16H26N2O4: C 61.91%, H 8.44%, N 9.03%, found: C
61.68%, H 8.37%, N 8.93%.
iodide (1.0 equiv instead of 0.5 equiv)
using 5 (471 mg, 3.00 mmol) and 1-
chloro-2-ethoxyethane
(651 mg,
6.00 mmol, 2.0 equiv). The title com-
pound was obtained as a yellow
solid (636 mg, 92%): mp: 37.2–
1
3
4
39.78C; H NMR (400 MHz, CDCl3): d=7.94 (dd, JH,H =9.2 Hz, JH,F
6.0 Hz, 1H, 6-H), 6.84 (dd, 3JH,F =10.3 Hz, 4JH,H =2.5 Hz, 1H, 3-H),
=
3
3
4
6.73 (ddd, JH,F =9.2 Hz, JH,H =7.1 Hz, JH,H =2.5 Hz, 1H, 5-H), 4.24 (t,
3JH,H =4.7 Hz, 2H, 1’-H2), 3.85 (t, 3JH,H =4.7 Hz, 2H, 2’-H2), 3.85 (q,
3JH,H =7.0 Hz, 2H, 4’-H2), 1.22 ppm (t, 3JH,H =7.0 Hz, 3H, 5’-H3);
13C NMR (126 MHz, CDCl3): d=165.8 (d, 1JC,F =256.7 Hz, 1C, C-4),
N-(3-Morpholinopropyl)-4-nitro-3-(pentyloxy)aniline (7v): Com-
pound 7v was prepared according to general procedure 2B using
6d (238 mg, 1.05 mmol) and 3-morpholinopropan-1-amine
(303 mg, 2.10 mmol, 2.0 equiv) with triethylamine (425 mg,
154.8 (d, 3JC,F =12.0 Hz, 1C, C-2), 136.1 (1C, C-1), 128.1 (3JC,F
12.0 Hz, 1C, C-6), 107.2 (2JC,F =22.8 Hz, 1C, C-5), 102.3 (2JC,F
=
=
26.4 Hz, 1C, C-3), 68.4 (1C, C-4’), 66.8 (1C, C-1’), 58.8 (1C, C-5’),
29.2 ppm (1C, C-2’); MS (ESI): m/z (%)=171 (10) [MÀC3H6O]+, 230
(100) [M+H]+, 247 (39) [M+NH4]+, 252 (21) [M+Na]+; HRMS (EI)
m/z [M]+ calcd for C10H12FNO4: 229.075036, found: 229.073714;
Anal. calcd for C10H12FNO4: C 52.40%, H 5.28%, N 6.11%, found: C
52.66%, H 5.47%, N 6.23%.
N-Hexyl-4-nitro-3-(pentyloxy)aniline (7d): Compound 7d was
prepared according to general procedure 2A using 6d (227 mg,
1.00 mmol) and hexan-1-amine (405 mg, 4.00 mmol, 4.0 equiv). The
title compound was obtained as a yellow solid (130 mg, 42%):
4.20 mmol, 4.0 equiv) as additional base. The title compound was
obtained as a yellow liquid (338 mg, 92%): 1H NMR (400 MHz,
CDCl3): d=7.96 (d, 3JH,H =9.2 Hz, 1H, 5-H), 6.10 (dd, 3JH,H =9.0 Hz,
3JH,H =2.4 Hz, 1H, 6-H), 6.00 (d, 2JH,H =2.3 Hz, 1H, 2-H), 4.04 (t,
3
3JH,H =6.5 Hz, 2H, 1’’-H2), 3.77 (t, JH,H =4.6 Hz, 4H, 6’-H2, 8’-H2), 3.30
(q, 3JH,H =5.8 Hz, 2H, 1’-H2), 2.57 (t, 3JH,H =6.2 Hz, 2H, 3’-H2), 2.54
(brs, 4H, 5’-H2, 9’-H2), 1.90–1.82 (m, 4H, 2’-H2, 2’’-H2), 1.54–1.46 (m,
2H, 3’’-H2), 1.39 (tq, 3JH,H =7.2 Hz, 3JH,H =7.2 Hz, 2H, 4’’-H2),
0.94 ppm (t, 3JH,H =7.2 Hz, 3H, 5’’-H3); 13C NMR (100 MHz, CDCl3):
d=156.7 (1C, C-3), 154.4 (1C, C-1), 129.3 (1C, C-5), 128.8 (1C, C-4),
104.1 (1C, C-6), 95.3 (1C, C-2), 69.4 (1C, C-1’’), 67.0 (2C, C-6’, C-8’),
57.6 (1C, C-3’), 53.7 (2C, C-5’, C-9’), 43.1 (1C, C-1’), 28.8 (1C, C-2’’),
28.1 (1C, C-3’’), 24.4 (1C, C-2’), 22.4 (1C, C4’’), 14.1 ppm (1C, C-5’’);
MS (ESI): m/z (%)=352 (100) [M+H]+, 374 (4) [M+Na]+; HRMS
(ESI) m/z [M+H]+ calcd for C18H30N3O4: 352.2236, found: 352.2231;
Anal. calcd for C18H29N3O4: C 61.52%, H 8.32%, N 11.96%, found: C
61.38%, H 8.32%, N 11.77%.
3
1H NMR (400 MHz, CDCl3): d=7.95 (d, JH,H =9.2 Hz, 1H, 5-H), 6.10
3
4
4
(dd, JH,H =9.2 Hz, JH,H =2.3 Hz, 1H, 6-H), 6.02 (d, JH,H =2.3 Hz, 1H,
2-H), 4.36 (brt, JH,H =5.0 Hz, 1H, NH), 4.04 (t, JH,H =6.5 Hz, 2H, 1’’-
H2), 3.17 (mc, 2H, 1’-H2), 1.90–1.83 (m, 2H, 2’’-H2), 1.64 (tt, JH,H
3
3
3
=
3
7.3 Hz, JH,H =7.3 Hz, 2H, 2’-H2), 1.53–1.31 (m, 10H, 3’-H2, 4’-H2, 5’-
H2, 3’’-H2, 4’’-H2), 0.93 (t, 3JH,H =7.2 Hz, 3H, 5’’-H3), 0.90 ppm (brt,
3JH,H =6.9 Hz, 3H, 6’-H3); 13C NMR (100 MHz, CDCl3): d=156.6 (1C,
C-3), 154.1 (1C, C-1), 129.3 (1C, C-5), 104.0 (1C, C-6), 95.7 (1C, C-2),
69.4 (1C, C-1’’), 43.5 (1C, C-1’), 31.6 (1C, C-2’), 29.2 (1C, C-4’), 28.8
(1C, C-2’’), 28.2 (1C, C-3’’), 26.8 (1C, C-3’), 22.7 (1C, C-5’), 22.5 (1C, C-
4’’), 14.1 ppm (2C, C-6’, C-5’’); MS (ESI): m/z (%)=221 (18)
[MÀC5H11O]+, 309 (100) [M+H]+, 331 (26) [M+Na]+, 617 (13)
[2M+H]+, 639 (15) [2M+Na]+, 925 (6) [3M+H]+; HRMS (ESI) m/z
[M+Na]+ calcd for C17H28N2O3Na: 331.199763, found: 331.199488.
3-(2-Ethoxyethoxy)-N-(3-morpholinopropyl)-4-nitroaniline (7w):
Compound 7w was prepared according to general procedure 2B
using 6m (229 mg, 1.00 mmol) and 3-morpholinopropan-1-amine
(288 mg, 2.00 mmol, 2.0 equiv) with triethylamine (405 mg,
3-(2-Ethoxyethoxy)-N-hexyl-4-nitroaniline (7u): Compound 7u
was prepared according to general procedure 2B using 6m
(229 mg, 1.00 mmol) and hexan-1-amine (202 mg, 2.00 mmol,
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4.00 mmol, 4.0 equiv) as additional base. The title compound was
obtained as a yellow liquid (343 mg, 97%): 1H NMR (400 MHz,
CDCl3): d=7.94 (d, 3JH,H =9.2 Hz, 1H, 5-H), 6.11 (dd, 3JH,H =9.0 Hz,
3JH,H =2.4 Hz, 1H, 6-H), 6.05 (d, 2JH,H =2.3 Hz, 1H, 2-H), 4.19 (t,
3JH,H =5.0 Hz, 2H, 1’’-H2), 3.86 (t, 3JH,H =5.0 Hz, 2H, 2’’-H2), 3.76 (t,
44.2 (1C, C-1’’), 31.7 (1C, C-4’’), 29.6 (1C, C-2’’), 28.7 (1C, C-2’’’), 28.0
(1C, C-3’’’), 26.9 (1C, C-3’’), 22.7 (1C, C-5’’), 22.5 (1C, C-4’’’), 14.1 (1C,
C-6’’), 14.1 ppm (1C, C-5’’’); MS (ESI): m/z (%)=278 (100)
[MÀC6H4O4S2]+, 483 (60) [M+H]+, 505 (58) [M+Na]+; HRMS (ESI)
m/z [M+H]+ calcd for C23H35N2O5S2: 483.198741, found:
483.201640; Anal. calcd for C23H34N2O5S2: C 57.24%, H 7.10%, N
5.80%, found: C 57.01%, H 7.10%, N 5.79%.
3
3JH,H =4.6 Hz, 4H, 6’-H2, 8’-H2), 3.65 (q, JH,H =7.0 Hz, 2H, 4’’-H2), 3.27
(q, 3JH,H =5.8 Hz, 2H, 1’’-H2), 2.56 (t, 3JH,H =6.3 Hz, 2H, 3’-H2), 2.53
3
3
(brs, 4H, 5’-H2, 9’-H2), 1.84 (tt, JH,H =6.2 Hz, JH,H =6.2 Hz, 2H, 2’-H2),
1.22 ppm (t, 3JH,H =7.0 Hz, 3H, 5’’-H3); 13C NMR (100 MHz, CDCl3):
d=156.5 (1C, C-3), 154.4 (1C, C-1), 129.2 (1C, C-5), 128.9 (1C, C-4),
104.7 (1C, C-6), 95.9 (1C, C-2), 69.4 (1C, C-2’’), 68.6 (1C, C-1’’), 67.2
(1C, C-4’’), 67.0 (2C, C-6’, C-8’), 57.6 (1C, C-3’), 53.7 (2C, C-5’, C-9’),
43.1 (1C, C-1’), 24.3 (1C, C-2’), 15.3 ppm (1C, C-5’’); MS (ESI): m/z
(%)=354 (100) [M+H]+, 376 (24) [M+Na]+; HRMS (ESI) m/z [M+
H]+ calcd for C17H28N3O5: 354.2023, found: 354.2029; Anal. calcd
for C17H27N3O5·0.25H2O: C 57.05%, H 7.74%, N 11.74%, found: C
57.21%, H 7.62%, N 11.54%.
Methyl
3-(N-(2-(2-ethoxyethoxy)-4-(hexylamino)phenyl)sulfa-
moyl)thiophene-2-carboxylate (9u): Compound 9u was prepared
according to general procedure 4 using 8u (0.500 mmol). The title
compound was obtained as an orange solid (79 mg, 33% over two
steps): mp: 80.8–81.58C; 1H NMR (400 MHz, CDCl3): d=8.20 (brs,
1H, SO2NH), 7.36 (d, 3JH,H =5.3 Hz, 1H, 5-H), 7.35 (d, 3JH,H =5.0 Hz,
N1-Hexyl-3-(pentyloxy)benzene-1,4-diamine (8d): 8d was pre-
pared following a slightly altered general procedure 3 using 7d
(105 mg, 0.340 mmol) and PtO2 (10 mg) as catalyst and a reaction
temperature of 408C. The title compound was obtained as
a purple solid which was directly used in the next step without fur-
ther purification.
1H, 4-H), 7.30 (d, 3JH,H =8.7 Hz, 1H, 6’-H), 6.14 (dd, 3JH,H =8.6 Hz,
3-(2-Ethoxyethoxy)-N1-hexylbenzene-1,4-diamine (8u): 8u was
prepared according to general procedure 3 using 7u (155 mg,
0.500 mmol). The title compound was obtained as a purple solid
which was directly used in the next step without further purifica-
tion.
4
4JH,H =2.4 Hz, 1H, 5’-H), 6.01 (d, JH,H =2.3 Hz, 1H, 3’-H), 3.98 (s, 3H,
3
3
CO2CH3), 3.88 (t, JH,H =5.4 Hz, 2H, 1’’’-H2), 3.55 (t, JH,H =5.5 Hz, 2H,
3
3
2’’’-H2), 3.51 (q, JH,H =7.1 Hz, 2H, 4’’’-H2), 3.00 (t, JH,H =7.1 Hz, 2H,
3
3
1’’-H2), 1.56 (tt, JH,H =7.2 Hz, JH,H =7.2 Hz, 2H, 2’’-H2), 1.39–1.26 (m,
3
6H, 3’’-H2, 4’’-H2, 5’’-H2), 1.22 (t, JH,H =7.0 Hz, 3H, 5’’’-H3), 0.88 ppm
(brt, JH,H =6.9 Hz, 3H, 6’’-H3); 13C NMR (100 MHz, CDCl3): d=160.7
3
N1-(3-Morpholinopropyl)-3-(pentyloxy)benzene-1,4-diamine (8v):
8v was prepared according to general procedure 3 using 7v
(176 mg, 0.500 mmol). The title compound was obtained as
a purple liquid which was directly used in the next step without
further purification.
(1C, CO2CH3), 152.2 (1C, C-2’), 147.6 (1C, C-4’), 145.3 (1C, C-3), 131.8
(1C, C-2), 131.3 (1C, C-5), 129.3 (1C, C-4), 127.1 (1C, C-6’), 115.2 (1C,
C-1’), 105.3 (1C, C-5’), 97.4 (1C, C-3’), 68.4 (1C, C-2’’’), 67.9 (1C, C-
1’’’), 66.8 (1C, C-4’’’), 53.0 (1C, CO2CH3), 44.2 (1C, C-1’’), 31.6 (1C, C-
4’’), 29.4 (1C, C-2’’), 26.8 (1C, C-3’’), 22.6 (1C, C-5’’), 15.2 (1C, C-5’’’),
14.1 ppm (1C, C-6’’); MS (ESI): m/z (%)=280 (100) [MÀC11H26NO2]+,
301 (10), 485 (34) [M+H]+, 507 (22) [M+Na]+; HRMS (ESI) m/z
[M+Na]+ calcd for C22H32N2O6S2Na: 507.159950, found:
507.157691; Anal. calcd for C22H32N2O6S2: C 54.52%, H 6.66%, N
5.78%, found: C 54.79%, H 6.70%, N 5.68%.
3-(2-Ethoxyethoxy)-N1-(3-morpholinopropyl)benzene-1,4-dia-
mine (8w): 8w was prepared according to general procedure 3
using 7w (177 mg, 0.500 mmol). The title compound was obtained
as a purple liquid which was directly used in the next step without
further purification.
Methyl 3-(N-(4-(hexylamino)-2-(pentyloxy)phenyl)sulfamoyl)thio-
phene-2-carboxylate (9d): Compound 9d was prepared according
to general procedure 4 using 8d (0.340 mmol). The title compound
was obtained as a yellow solid (84 mg, 51% over two steps): mp:
80.4–82.98C; 1H NMR (400 MHz, CDCl3): d=8.18 (brs, 1H, SO2NH),
Methyl 3-(N-(4-((3-morpholinopropyl)amino)-2-(pentyloxy)phe-
nyl)sulfamoyl)thiophene-2-carboxylate (9v): Compound 9v was
prepared according to general procedure 4 using 8v (0.500 mmol).
The title compound was obtained as a yellow solid (134 mg, 51%
over two steps): mp: 93.9–96.78C; 1H NMR (400 MHz, CDCl3): d=
3
3
3
3
7.38 (d, JH,H =5.3 Hz, 1H, 5-H), 7.36 (d, JH,H =5.3 Hz, 1H, 4-H), 7.32
8.19 (s, 1H, SO2NH), 7.38 (d, JH,H =5.4 Hz, 1H, 5-H), 7.36 (d, JH,H
=
=
3
3
4
3
5.2 Hz, 1H, 4-H), 7.31 (d, 3JH,H =8.6 Hz, 1H, 6’-H), 6.11 (dd, JH,H
(d, JH,H =8.7 Hz, 1H, 6’-H), 6.12 (dd, JH,H =8.6 Hz, JH,H =2.4 Hz, 1H,
4
2
2
5’-H), 5.94 (d, JH,H =2.5 Hz, 1H, 3’-H), 3.97 (s, 3H, CO2CH3), 3.68 (t,
8.6 Hz, JH,H =2.3 Hz, 1H, 5’-H), 5.95 (d, JH,H =1.4 Hz, 1H, 3’-H), 4.63
(brs, 1H, NH), 3.97 (s, 3H, CO2CH3), 3.77 (brs, 4H, 6’’-H2, 8’’-H2), 3.67
3JH,H =7.0 Hz, 2H, 1’’’-H2), 3.54 (brs, 1H, NH), 3.02 (t, JH,H =7.2 Hz,
3
(t, 3JH,H =7.0 Hz, 2H, 1’’’-H2), 3.15 (t, 3JH,H =5.9 Hz, 2H, 1’’-H2), 2.55
2H, 1’’-H2), 1.56 (mc, 4H, 2’’-H2, 2’’’-H2), 1.40–1.28 (m, 10H, 3’-H2, 4’’-
3
3
H2, 5’’-H2, 3’’’-H2, 4’’’-H2), 0.92 (t, JH,H =7.1 Hz, 3H, 5’’’-H3), 0.89 ppm
(brs, 6H, 3’’-H2, 5’’-H2, 9’’-H2), 1.82 (brs, 2H, 2’’’-H2), 1.56 (tt, JH,H
=
(brt, 3JH,H =7.1 Hz, 3H, 6’’-H3); 13C NMR (100 MHz, CDCl3): d=60.7
(1C, CO2CH3), 152.5 (1C, C-2’), 147.9 (1C, C-4’), 145.9 (1C, C-3), 131.5
(1C, C-2), 131.4 (1C, C-5), 129.3 (1C, C-4), 127.4 (1C, C-6’), 114.8 (1C,
C-1’), 104.4 (1C, C-5’), 96.5 (1C, C-3’), 68.2 (1C, C-1’’’), 53.0 (CO2CH3),
7.2 Hz, 3JH,H =7.2 Hz, 2H, 2’’-H2), 1.36–1.27 (m, 4H, 3’’’-H2, 4’’’-H2),
0.92 ppm (t, 3JH,H =7.0 Hz, 3H, 5’’’-H3); 13C NMR (100 MHz, CDCl3):
d=160.6 (1C, CO2CH3), 152.5 (1C, C-2’), 147.9 (1C, C-4’), 145.8 (1C,
C-3), 131.5 (1C, C-2), 131.3 (1C, C-5), 129.4 (1C, C-4), 127.4 (1C, C-6’),
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114.7 (1C, C-1’), 104.3 (1C, C-5’), 96.5 (1C, C-3’), 68.2 (2C, C-6’’, C-8’’),
66.9 (1C, C-1’’’), 57.5 (1C, C3’’), 53.7 (2C, C-5’’, C-9’’), 53.0 (1C,
CO2CH3), 43.2 (1C, C-1’’), 28.7 (1C, C-2’’’), 27.9 (1C, C-3’’’), 25.2 (1C,
C-2’’), 22.5 (1C, C-4’’’), 14.1 ppm (1C, C-5’’’); MS (ESI): m/z (%)=321
(21) [MÀC6H6O4S2]+, 526 (100) [M+H]+; Anal. calcd for
C24H35N3O6S2: C 54.84%, H 6.71%, N 7.99%, found: C 54.93%, H
6.65%, N 8.08%.
TR-FRET: Interaction of the PPARb/d LBD with co-repressor peptides
was determined using the Lanthascreen TR-FRET PPARb/d co-regu-
lator assay with a fluorescein-labeled co-repressor peptide derived
from the SMRT-interaction domain 2.[17] Measurements were per-
formed on a VICTOR3V Multilabel Counter (WALLAC 1420; Perki-
nElmer, Waltham, MA, USA).
IC50 calculations: IC50 values were determined by nonlinear regres-
sion analysis using Prism version 6.0 software (GraphPad Software,
San Diego, CA, USA).
Methyl 3-(N-(2-(2-ethoxyethoxy)-4-((3-morpholinopropyl)amino)-
phenyl)sulfamoyl)thiophene-2-carboxylate (9w): Compound 9w
was prepared according to general procedure 4 using 8w
(0.500 mmol). The title compound was obtained as a yellow solid
Quantitative RT-PCR: RNA isolation was carried out according to the
manufacturer’s instructions using the NucleoSpin RNA Isolation Kit
(Macherey–Nagel GmbH & Co. KG, Düren, Germany). cDNA was
synthesized from 500 ng of RNA according to the manufacturer’s
protocol with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories
GmbH, Munich, Germany). Real-time quantitative PCR was per-
formed in triplicate reactions in an Mx3000P RT-qPCR system (Stra-
tagene, La Jolla, CA, USA) using ABsolute QPCR SYBR Green Mix
(Thermo Fisher Scientific, Waltham, MA, USA) at a primer concen-
tration of 0.25 mm. RPL27/Rpl27 primers were used for normaliza-
tion.
1
(104 mg, 39% over two steps): H NMR (400 MHz, CDCl3): d=8.20
3
3
(s, 1H, SO2NH), 7.37 (d, JH,H =5.2 Hz, 1H, 5-H), 7.36 (d, JH,H =5.2 Hz,
Human primers: RPL27 forward: AAA GCT GTC ATC GTG AAG AAC,
RPL27 reverse: GCT GTC ACT TTG CGG GGG TAG, PDK4 forward:
TTA TAC ATA CTC CAC TGC ACC A, PDK4 reverse: ATA GAC TCA
GAA GAC AAA GCC T.
1H, 4-H), 7.30 (d, 3JH,H =8.6 Hz, 1H, 6’-H), 6.13 (dd, 3JH,H =8.6 Hz,
2JH,H =2.6 Hz, 1H, 5’-H), 6.00 (d, 2JH,H =2.3 Hz, 1H, 3’-H), 4.62 (brs,
3
1H, NH), 3.99 (s, 3H, CO2CH3), 3.88 (t, JH,H =5.3 Hz, 2H, 1’’’-H2), 3.77
(brs, 4H, 6’’-H2, 8’’-H2), 3.56 (t, 3JH,H =5.3 Hz, 2H, 2’’’-H2), 3.52 (q,
Mouse primers: Rpl27 forward: AAA GCC GTC ATC GTC AAC AAC,
Rpl27 reverse: GCT GTC ACT TTC CCG GGG ATA G, Angptl4 for-
ward: CTC TGG GGT CTC CAC CAT TT, Angptl4 reverse: TTG GGG
ATC TCC GAA GCC AT, Cpt1a forward: AGT GTG TGA GGC CAC
TGA TG, Cpt1a reverse: TCG CTG CCT GAA TAT GGG TT.
3
3JH,H =7.0 Hz, 2H, 4’’’-H2), 3.13 (t, JH,H =6.3 Hz, 2H, 1’’-H2), 2.54 (brs,
3
3
6H, 3’’-H2, 5’’-H2, 9’’-H2), 1.81 (tt, JH,H =6.3 Hz, JH,H =6.3 Hz, 2H, 2’’-
H2), 1.22 ppm (t, 3JH,H =7.0 Hz, 3H, 5’’’-H3); 13C NMR (100 MHz,
CDCl3): d=160.7 (1C, CO2CH3), 152.3 (1C, C-2’), 147.9 (1C, C-4’),
145.4 (1C, C-3), 131.8 (1C, C-2), 131.3 (1C, C-5), 129.3 (1C, C-4),
127.2 (1C, C-6’), 115.3 (1C, C-1’), 105.1 (1C, C-5’), 97.4 (1C, C-3’), 68.5
(1C, C-2’’’), 68.0 (1C, C-1’’’), 66.8 (1C, C-4’’’), 66.7 (2C, C-6’’, C-8’’),
57.4 (1C, C-3’’), 53.6 (2C, C-5’’, C-9’’), 53.1 (1C, CO2CH3), 43.1 (1C, C-
1’’), 25.1 (1C, C-2’’), 15.2 ppm (1C, C-5’’’); MS (ESI): m/z (%)=323
(37) [MÀC6H6O4S2]+, 528 (100) [M+H]+; HRMS (ESI) m/z [M+H]+
calcd for C23H34N3O7S2: 528.1838, found: 528.1833; Anal. calcd for
C23H33N3O7S2·0.25H2O: C 51.91%, H 6.35%, N 7.90%, found: C
51.79%, H 6.21%, N 7.97%.
Pharmacokinetics in mice: Compounds were administered intrave-
nously (i.v.) at 1 mgkgÀ1 in 200 mL PBS/Solutol 5% to male mice
(six months of age, weight: ~25 g, three animals per data point),
and blood samples were taken at the indicated time. Plasma sam-
ples (50 mL) were diluted with acetonitrile (200 mL) and centrifuged
for 15 min at 48C (6000g); 180 mL of the supernatant were diluted
with water (100 mL) and again centrifuged (15 min, 3900g); 200 mL
of the resulting supernatant were analyzed further by HPLC–MS/
MS. The measurements were carried out on a Waters Alliance sepa-
ration system (Model 2795, Milford, MA, USA). Samples were sepa-
rated on a Phenomenex Kinetex column (C18, length: 50 mm, inner
diameter: 2.1 mm, particle size: 2.6 mm, pore size: 100 Š) using iso-
cratic conditions (80% MeCN, 20% 10 mm NH4OAc, flow rate:
0.2 mLminÀ1, injection volume: 10 mL, run time: 8 min). The separa-
tion system was coupled to a nano-electrospray interface of a Mi-
cromass Quattro Micro mass spectrometric detector (Beverly, MA,
USA). The compounds were detected in multiple reaction monitor-
ing mode. For quantification, a ten-point calibration curve was
used with the corresponding transitions (9u: m/z=485!m/z=
280). Data analysis was performed using Masslynx software Ver-
sion 4.1 (Waters, Milford, MA, USA). The coefficient of determina-
tion (R2) was better than 0.99. Limit of quantification (LOQ) for 9u:
Biological data
Mice: C57 BL6 mice were purchased from Charles River Laborato-
ries (Wilmington, MA, USA). Mouse experiments were approved by
the local authorities (Regierungspräsidium Giessen).
Cell culture: C2C12 murine myoblasts (American Type Culture Col-
lection, Manassas, VA, USA) were cultured in Dulbecco’s modified
Eagle’s medium (DMEM), complemented with 10% fetal calf serum
(FCS), 2 mm l-glutamine, 100 UmLÀ1 penicillin, 100 mgmLÀ1 strep-
tomycin. MDA-MB-231-luc2 (Caliper Life Science, Mainz, Germany)
human breast cancer cells were cultured in McCoy’s 5A (Modified)
medium complemented with 10% FCS, 100 UmLÀ1 penicillin,
100 mgmLÀ1 streptomycin. Cells were maintained in a humidified
incubator at 378C and 5% CO2. Cells were exposed to ligands
(1 mm or indicated concentrations) for 24 h. Mononuclear cells
were isolated from peripheral blood of healthy volunteers by Lym-
phocyte Separation Medium 1077 (PromoCell GmbH, Heidelberg,
Germany) density gradient centrifugation followed by adherent
cell positive selection. Cells were cultured in RPMI 1640 supple-
mented with 10% FCS for six days. Ligand treatment was per-
formed as indicated at 300 nm final concentrations.
0.005 ngmLÀ1; limit of detection (LOD) for 9u: 0.0015 ngmLÀ1
.
Acknowledgements
We gratefully acknowledge the financial support of this work by
a grant from the Deutsche Forschungsgemeinschaft (DFG) to
W.E.D. (DI 827/4-1) and T.A. (AD 474/1-1).
ChemMedChem 2016, 11, 488 – 496
www.chemmedchem.org
495
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Received: December 23, 2015
Published online on February 11, 2016
ChemMedChem 2016, 11, 488 – 496
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496
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim